1. Field of the Invention
The present invention relates to the production of recombinant polyclonal proteins, such as proteins from the immunoglobulin superfamily, e.g. soluble or membrane-bound forms of B or T cell receptors, using production systems which are independent of site-specific integration.
2. Background Art
A number of diseases such as infectious diseases and cancers lack efficient therapies. Monoclonal antibodies have generally not been successful against all of these targets, partly due to variability of the complex targets and adaptive mutations of target proteins causing immune escape from monoclonal antibody recognition. Polyclonal antibodies on the other hand are able to target a plurality of dynamic targets, e.g., on viruses, bacteria, or cancer cells. Also, polyclonal antibodies have the highest probability of retaining activity in the event of antigenic mutation.
Different commercially available polyclonal antibody therapeutics exist including: 1) normal human immunoglobulin isolated from the blood of normal human donors; 2) human hyperimmune immunoglobulin derived from the blood of individual human donors carrying antibodies against a particular disease target, e.g., a virus, which they previously have encountered either through infection or vaccination; and 3) animal hyperimmune immunoglobulin derived from the blood of immunized animals.
Immunoglobulin purified from human blood has proved effective against infections with hepatitis B virus, respiratory syncytial virus, cytomegalovirus and other herpes viruses, rabies virus, botulinum toxin, etc, as well as in the neonatal rhesus D prophylaxis. Immunoglobulin purified from the blood of rabbits immunized with human T cells is used to afford T cell immunosuppression in the treatment or prevention of transplant rejection (e.g., Thymoglobulin). Normal human immunoglobulin has been utilized to boost the immune system of immunodeficient patients, as well as in the therapy of various autoimmune disorders.
Nevertheless, widespread immunoglobulin use has been limited due to the constrained supply of donor blood raw material, problems with batch-to-batch variations, and variable safety. Animal-derived immunoglobulins in particular are faced with the same problems of immunogenicity as was observed for animal-derived monoclonal antibodies in the 1980s and 1990s. Finally, as with other blood products, the risk of transmission of infectious agents such as HIV, herpes or hepatitis viruses or prions remains. Accordingly, while clinicians acknowledge that polyclonal antibodies are a preferred therapeutic in some situations, their use has been very limited.
New approaches to generate human immunoglobulins arose with the transgenic animal techniques. Transgenic mice carrying human immunoglobulin loci have been created (U.S. Pat. No. 6,111,166). These mice produce fully human immunoglobulins, and antibodies against a specific target can be raised by usual immunization techniques. However, larger antibody yields are limited because of the relatively small size of mice. Larger animals have also been made transgenic for the human immunoglobulin genes, e.g., cows (Kuroiwa, Y. et al. Nature Biotechnology; 2002; 20: 889-893). However, producing polyclonal antibodies for therapy from the blood of such animals is not without complications. First, the immunophysiology of the animal and humans may display considerable differences, causing a difference in the resulting immune repertoire, functional rearrangement, and diversity of the antibody response. Second, mitotic instability of the introduced immunoglobulin loci might influence the long-term production of antibodies. Third, it is technically challenging to delete the animal's own immunoglobulin loci so that e.g., the animal antibody production will not exceed the production of human antibody. Fourth, the risk of transmission of infectious agents such as viruses, prions or other pathogens accompanies the administration of human antibodies produced in animals.
Recently, a new type of polyclonal antibodies which is independent on donor availability at the time of production has been developed. These polyclonal antibodies are generated by isolating antibody encoding nucleic acid sequences from donors with an immune response against the desired target, followed by screening for antibodies which specifically bind the desired target. The polyclonal antibody may be manufactured by an adapted mammalian expression technology, which is based on site-specific integration of one antibody expression plasmid into the same genomic site of each cell as described in WO 2004/061104. One example of this new type of polyclonal antibodies is a recombinant polyclonal antibody against Rhesus D (WO 2006/007850). The use of site-specific integration results in a cell population where each cell contains a single copy and where expression levels and growth rates are expected to be relatively uniform.